In the present protocol, the endothelial barrier function of the submandibular gland (SMG) was evaluated by injecting different molecular weighted fluorescent tracers into the angular veins of test animal models in vivo under a two-photon laser-scanning microscope.
Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the endothelial barrier dysfunction is related to many salivary gland secretory disorders. The present protocol describes an in vivo paracellular permeability detection method to evaluate the function of endothelial tight junctions (TJs) in mouse submandibular glands (SMG). First, fluorescence-labeled dextrans with different molecular weights (4 kDa, 40 kDa, or 70 kDa) were injected into the angular veins of mice. Afterward, the unilateral SMG was dissected and fixed in the customized holder under a two-photon laser-scanning microscope, and then images were captured for blood vessels, acini, and ducts. Utilizing this method, the real-time dynamic leakage of the different-sized tracers from blood vessels into the basal sides of the acini and even across the acinar epithelia into the ducts was monitored to evaluate the alteration of the endothelial barrier function under physiological or pathophysiological conditions.
Various salivary glands produce saliva, which primarily acts as the first line of defense against infections and helps digestion, thereby playing an essential role in oral and overall health1. Blood supply is crucial for salivary gland secretion since it constantly provides water, electrolytes, and molecules that form the primary saliva. Endothelial barrier function, regulated by the tight junction (TJ) complex, strictly and delicately limits the permeation of capillaries, which are highly permeable to water, solutes, proteins, and even cells moving from the circulating blood vessels into the salivary gland tissues2,3. We have previously found that the opening of the endothelial TJs in response to a cholinergic stimulus facilitates saliva secretion, whereas the impairment of the endothelial barrier function is interlinked with hyposecretion and lymphocytic infiltration in the submandibular glands (SMGs) in Sjögren's syndrome4. These data suggest that the contribution of endothelial barrier function needs to be paid enough attention regarding a variety of salivary gland diseases.
A two-photon laser-scanning microscope is a powerful tool for observing the dynamics of cells in intact tissue in vivo. One of the advantages of this technique is that near-infrared light (NIR) has deeper tissue penetration than visible or ultraviolet light when specimens are excited by NIR and does not cause obvious light damage to tissues under appropriate conditions5,6. Indeed, the salivary glands are a very homogenous and superficial tissue, in which the surface acinar cells are only around 30 µm away from the gland surface7,8. It has been shown that intravital confocal microscopy can study exocrine secretion and actin cytoskeleton in live mouse salivary glands at subcellular resolution8. Two-photon laser-scanning microscopy, nevertheless, not only has the advantage of conventional confocal microscopy but can also be used to detect deeper tissue and image more clearly. Here, fluorescence-labeled dextrans, which are frequently used as paracellular permeability tracers and have the advantage of different sizes, can be used to test the magnitude of TJ pore9. In the present study, an intravital real-time two-photon laser-scanning microscopy technique is established for in situ evaluation of endothelial barrier function in mouse SMGs. Each work step for in vivo vascular permeability detection in mouse SMGs is described in the current protocol. Here is an example of detecting endothelial barrier function in the mouse SMG duct ligation model.
All experimental procedures were approved by the Ethics Committee of Animal Research, Peking University Health Science Center, and complied with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996). Male wild-type (WT) mice in the age group of 8-10 weeks were used for the present study. The experimental animals were carefully treated to minimize their pain and discomfort.
1. Animal procedures
2. Two-photon microscope set-up
3. Vessel imaging and permeability detection
4. Data analysis
5. Downstream applications
6. Animal care and recovery
Following the protocol, the unilateral SMG was attached to a custom-made holder, and the gland was kept as far away from the mouse body as possible to prevent breathing from causing motion artifacts. The rapid flow of the red blood cells (black dots) in blood vessels was observed under the microscope. After finding the tissue field under an ocular lens, one must switch to manipulate the microscope software. In the control group, both the tracers existed in the blood vessels of the mouse SMG. In particular, due to its small molecular weight, FD4 was able to leak out of the blood vessels to the basal sides of acini and ducts, thereby clearly depicting the shape of the acini and ducts (as A and D point out in Figure 2A). By contrast, dextrans with higher molecular weights, such as 40 kDa and 70 kDa, could not differentiate the SMG morphology. Indeed, RD70 was dominantly distributed in large-sized blood vessels and microvessels. In the duct ligation group, both FD4 and RD70 were extravasated to the basal sides of acini, which indicated that duct ligation could disrupt the endothelial barrier function and then increase the permeability to large molecules. The semi-quantitative FD4 and RD70 fluorescence intensity results also confirmed the above phenomena (Figure 2B). Besides, the diameter of the blood vessels was increased, indicating that duct ligation for 1 day induced dilation of blood vessels. Furthermore, the 3D images showed much more obscured fluorescence of FD4 and RD70 around blood vessels in the ligation group (Figure 2C).
Figure 1: A schematic diagram showing the customized holder. The holder is shaped like a miniature magnifying glass with a flat piece of round glass in the center (diameter: 4.32 mm). One side of the holder is connected with the negative pressure device to suck up the tissues under the glass, while the other side of the holder is dead. Please click here to view a larger version of this figure.
Figure 2: In vivo vascular permeability assay and 3D images of blood vessels in mice submandibular glands (SMGs). The mice were divided into control and duct ligation groups. The unilateral glands in both groups were exposed and observed. (A) The in vivo vascular permeability assay was performed by injecting 4 kDa FITC-labeled dextran (FD4) and 70 kDa rhodamine B-labeled dextran (RD70) into the angular vein. Arrowheads indicate the changes in the distribution of tracers in the SMG. A, acini. D, duct. Scale bar = 100 µm. (B) For the semi-quantification analysis of the above images, the fluorescence intensity, including FD4 and RD70 within blood vessels (intravascular) and outside blood vessels (extravascular), was measured by the Image J software. (C) 3D images of blood vessels and microvessels in SMG. Arrowheads indicate the changes in the distribution of tracers in the SMG. Please click here to view a larger version of this figure.
The maintenance and regulation of endothelial barrier function are essential for vascular homeostasis. Endothelial cells and their intercellular junctions play a critical role in maintaining and controlling vascular integrity12. The shear force of blood flow, growth factors, and inflammatory factors can cause changes in vascular permeability and, thus, participate in the occurrence and development of systemic diseases such as hypertension, diabetes, and autoimmune diseases13,14,15. The vascular distribution and blood flow of the salivary gland is one of the most abundant in all organs and tissues, but research on the vascular system of the salivary gland has not been carried out widely. A deeper and more comprehensive understanding of salivary gland blood vessels, particularly the endothelial barrier function, will provide new clues to the mechanism of salivary secretion and promote vascular biology research.
The in vivo two-photon laser-scanning microscopy technique can quantify the vascular permeability by intravenous injection of fluorescent dye without sacrificing the animal. Fluorescence-labeled dextrans with microspheres of different molecular weights or sizes can better assess the extent of endothelial injury. Meanwhile, the dynamic kinetics of vascular permeability are determined in real-time by using the intravital evaluation system of vascular permeability. Another advantage is that it is easy to distinguish the types of blood vessels, such as arterioles, venules, and capillaries16. Furthermore, destruction of the vascular endothelial barrier and migration of immune cells are inevitably linked to inflammation, but there are many studies investigating a single factor, and only a few studies have focused on the connection between both aspects17,18. Uhl et al. recently established a research method to analyze the role of different pathogenic factors in salivary gland diseases by injecting fluorescence-labeled dextrans and immune cell-specific antibodies with different fluorescent markers simultaneously in vivo19. Here, fluorescence-labeled immune cells can also be traced through tail vein injection to explore pathogenesis in the current working model. Therefore, the present experimental method may provide a unique system for studying the interaction between leukocyte extravasation and endothelial barrier integrity in SMG disease animal models in vivo.
Nevertheless, it must not be ignored that the responsiveness of mice to anesthetics is different, and the longer the imaging time takes, the longer the anesthesia time needed, and the more difficult it is for mice to recover. Therefore, this experiment is better used to investigate when no subsequent in vivo experiments are required after the imaging. In addition, another limitation is that this technique is not suitable for experiments with a long time span. Dextrans are water-soluble and easy to metabolize, resulting in weaker fluorescence with a longer imaging time, and thus, it is better to maintain imaging time within 30 min.
Altogether, the study focuses on the penetration of paracellular permeability tracers and cells from blood vessels and microvessels into glandular tissues and has rigorously established contrast-enhanced intravital dynamic two-photon laser-scanning microscopy as an advanced method to measure vascular permeability to evaluate endothelial barrier function in the mouse SMG.
The authors have nothing to disclose.
This study was supported by the National Natural Science Foundation of China (grants 31972908, 81991500, 81991502, 81771093, and 81974151) and the Beijing Natural Science Foundation (grant 7202082).
2-photon microscope (TCS-SP8 DIVE) | Leica, Germany | ||
4 kDa FITC-labeled dextran | Sigma Aldrich | 46944 | |
70 kDa rhodamine B-labeled dextran | Sigma Aldrich | R9379 | |
Blunt tissue separation nickel | Bejinghuabo Company | NZW28 | |
Depilatory cream | Veet | ||
Disposable sterile syringe | Zhiyu Company | 1 mL | |
Image J software | National Institutes of Health | ||
Insulin syringe | Becton, Dickinson and Company | 0253316 | 1 mL |
Leica Application Suite X software | Leica Microsystems | ||
Microtubes | Axygen | MCT-150-C | 1.5 mL |
Phosphate buffered saline 1x | Servicebio | G4207-500 | |
Tissue scissors | Bejinghuabo Company | M286-05 | |
Tribromoethanol | JITIAN Bio | JT0781 |